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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6635 | |||||||||
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Title | Glutamate dehydrogenase in complex with NADH, open conformation | |||||||||
![]() | Reconstruction of the BINARY complex of bovine glutamate dehydrogenase with NADH. This entry is the OPEN conformation. | |||||||||
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![]() | enzyme / glutamate metabolism / mitochondria | |||||||||
Function / homology | ![]() glutamate dehydrogenase [NAD(P)+] activity / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / : / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / glutamate catabolic process / amino acid metabolic process / glutamine metabolic process / oxidoreductase activity ...glutamate dehydrogenase [NAD(P)+] activity / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / : / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / glutamate catabolic process / amino acid metabolic process / glutamine metabolic process / oxidoreductase activity / mitochondrial inner membrane / mitochondrial matrix / nucleotide binding / GTP binding / endoplasmic reticulum / mitochondrion / ATP binding / identical protein binding Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
![]() | Borgnia MJ / Banerjee S / Merk A / Matthies D / Bartesaghi A / Rao P / Pierson J / Earl LA / Falconieri V / Subramaniam S / Milne JLS | |||||||||
![]() | ![]() Title: Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase. Authors: Mario J Borgnia / Soojay Banerjee / Alan Merk / Doreen Matthies / Alberto Bartesaghi / Prashant Rao / Jason Pierson / Lesley A Earl / Veronica Falconieri / Sriram Subramaniam / Jacqueline L S Milne / ![]() Abstract: Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping ...Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.5 KB 14.5 KB | Display Display | ![]() |
Images | ![]() | 243 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 408.6 KB | Display | ![]() |
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Full document | ![]() | 408.1 KB | Display | |
Data in XML | ![]() | 6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3jd2MC ![]() 6630C ![]() 6631C ![]() 6632C ![]() 6633C ![]() 6634C ![]() 3jczC ![]() 3jd0C ![]() 3jd1C ![]() 3jd3C ![]() 3jd4C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Reconstruction of the BINARY complex of bovine glutamate dehydrogenase with NADH. This entry is the OPEN conformation. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.676 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Binary complex of bovine glutamate dehydrogenase with NADH
Entire | Name: Binary complex of bovine glutamate dehydrogenase with NADH |
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Components |
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-Supramolecule #1000: Binary complex of bovine glutamate dehydrogenase with NADH
Supramolecule | Name: Binary complex of bovine glutamate dehydrogenase with NADH type: sample / ID: 1000 Details: The sample was largely monodisperse. Some chains of hexamers were observed. Image processing revealed the presence of two conformational states (see related entry EMD-6634). Oligomeric state: one homohexamer of GDH binds 12 molecules of NADH Number unique components: 2 |
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Molecular weight | Theoretical: 344 KDa |
-Macromolecule #1: L-glutamate:NAD(P)+ oxidoreductase (deaminating)
Macromolecule | Name: L-glutamate:NAD(P)+ oxidoreductase (deaminating) / type: protein_or_peptide / ID: 1 Name.synonym: Glutamate dehydrogenase 1, mitochondrial, glutamate dehydrogenase [NAD(P)+], GDH Details: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion ...Details: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column. Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 56 KDa |
Sequence | UniProtKB: Glutamate dehydrogenase 1, mitochondrial GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, ...GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, mitochondrion, mitochondrial inner membrane, mitochondrial matrix, amino acid metabolic process, glutamate catabolic process, glutamine metabolic process, oxidoreductase activity, oxidoreductase activity, GO: 0055114, GO: 0055114, tricarboxylic acid metabolic process InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation ...InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain, NAD(P)-binding domain |
-Macromolecule #2: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]me...
Macromolecule | Name: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate type: ligand / ID: 2 / Name.synonym: NADH, Nicotinamide Adenine Dinucleotide / Number of copies: 12 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 1 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 2.0 mg/mL |
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Buffer | pH: 6.8 Details: 100 mM potassium phosphate, 0.1% n-octyl glucopyranoside, 20 mM NADH |
Grid | Details: 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3-6 seconds before plunging. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: Gatan GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | Nov 23, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1588 / Average electron dose: 45 e/Å2 Details: Every image is the average of 38 frames recorded by the direct electron detector. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 73964 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
CTF correction | Details: Each micrograph |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2, Relion / Number images used: 34716 |